Dynamic cellular interactions that determine place coding in the auditory systems, termed tonotopy, are poorly understand. We propose to observe the formation of specialized tonotopic neural projection in vitro and in vivo. In doing so, we will develop a uniquely powerful model system for study of topographically organized innervation patterns, and provide information useful for designing strategies to reconnect axons that are severed by accidental damage, or cell populations that are denervated by surgical interventions, genetic anomalies, or disease. Our long term goals are to understand the cellular and molecular mechanisms that direct selective formation, stabilization and maturation of circuit-specific synaptic connections, and how such mechanisms can be modulated to offer opportunities to repair damaged nervous system components. The objectives of this application, which are the first steps in the pursuit of these goals, are to determine the timing and structural features of fundamental events that lead to tonotopic innervation. In Aim 1 we will determine the temporal and spatial dynamics of the outgrowth of globular bushy cell axons into the MNTB using organotypic brainstem slice cultures and fixed tissue in situ preparations: In Aim 2 we will reveal the extent to which growth cone behaviors are guided by intrinsic or extrinsic mechanisms, by establishing normal and ectopic co- cultures of embryonic brainstem explants to study the maturational effects of diffusible guidance signals in conferring proper pathfinding in globular bushy cell axons. Aim 3 will characterize the sequence of contact- mediated events resulting in the formation of a synaptic calyx. We will distinguish between several possible mechanisms of directed axonal outgrowth that may affect for calyceal innervation of the MNTB. Our Aim 4 is to reveal the reorganization of axonal ultrastructure underlying cochlear nucleus axonal branch sites. The proposed research combines comprehensive light and electron microscopic imaging techniques to uncovered the temporal, spatial, and morphological features associated with axonal pathfinding in a CNS circuit in vivo. We expect that the proposed research will make fundamental new contributions to the field of developmental biology in general and to auditory neurobiology to particular.